Degenerative conditions of large weight bearing joints resulting from aging and/or pathologic conditions such as arthritis and osteoporosis lead to surgical intervention approaches chiefly total joint replacement (TJR). However, implant-derived wear debris occurs with time causing inflammatory responses culminating with osteolysis and failure of implants. Subsequent revision surgery of the failing joint implant, is often more difficult, and associated with increased morbidity and mortality especially for aging patients with weaker bones. Therefore, the need for effective approaches to diagnose, prevent, and/or treat complications of TJR have risen in recent years. Thus, better understanding of the processes and mechanisms underlying pathologic and osteolytic events leading to joint failure is essential to provide appropriate preventive and therapeutic countermeasures. The pathologic response to implant wear-debris constitutes a major component of this phenomenon and is under intense investigation. Recent work by several groups including ours has identified important cellular entities and secreted factors that contribute to inflammatory osteolysis. In previous work, we have shown that PMMA particles contribute to inflammatory osteolysis through stimulation of major pathways in osteoclast precursors, primarily NF-?B and MAP kinases. The former pathway requires assembly of large IKK complex encompassing IKK1, IKK2, and IKK?, also known as NEMO. We have shown recently that interfering with the NF-?B and MAPK activation pathways, through introduction of inhibitors and decoy molecules, impede PMMA-induced osteolysis in mouse models of experimental calvarial osteolysis and inflammatory arthritis. In our recent work, we found that PMMA particles directly activate the upstream transforming growth factor beta activated kinase-1 (TAK1) which is a key regulator of signal transduction cascades leading to activation of NF-?B and AP-1 factors. More importantly, we found that PMMA particles induce TAK1 binding to NEMO, RIP1, and UBC13. In addition, we show that PMMA particles induced TRAF6 binding to NEMO and lack of TRAF6 significantly attenuates NEMO ubiquitination. We further demonstrate that PMMA induction of NF-?B and MAPK is impaired in TAK1-null and NEMO mutant cells. These responses were not aided by TNF or RANKL. Altogether, these results led us to hypothesize that PMMA particles maybe inducing K63-linked ubiquitination of NEMO, RIP1, and other target proteins, events likely mediated by TRAF6, TAK1 and UBC13. Relevant to this hypothesis, it has been documented that a key mediator of LPS, IL-1, and RANKL signaling, namely TRAF6, is ubiquitin ligase. In separate studies, it was further established that a variety of upstream signals augment ubiquitination-based signaling network dominated by TAK1/TABs/RIP/NEMO/UBC complex. Thus, we propose to investigate the following specific aims: 1. Delineate the molecular steps underlying PMMA-induced regulation of NEMO. 2. Investigate if TRAF6/TAK1/NEMO POLYUBIQUITINATION events mediate PMMA-induced osteoclastogenesis. 3. Determine the effect of inhibiting POLY-UB-NEMO signaling on PMMA-induced calvarial osteolysis. PUBLIC HEALTH RELEVANCE: Total joint implant failure is attributed, at least in part, to orthopedic particle-induced osteolysis. The mechanisms underlying this pathologic condition remain unclear. Our recent work has implicated heightened osteoclast activity, owing to enhanced intracellular activation of NF-?B and MAP kinase pathways, as the leading cause for inflammatory osteolysis. We provide evidence that key mediators of these two pathways, including TGF2-activating kinase (TAK1), IKK?/NEMO, E2-ligases such as Ubc13, and key ubiquitin-dependent events are induced by polymethylmethacrylate (PMMA) particles in osteoclast precursors. Thus, we will utilize in vitro and in-vivo approaches including genetically-modified mice to examine the role of ubiquitin-mediated events that contribute to exacerbation of PMMA-induced inflammatory osteolysis. Our proposal addresses novel questions and holds promise to identify novel selective anti-osteolytic therapies.